The goal of our studies of T lymphocytes is to identify novel and functionally important proteins (enzymes, receptors) and to find their specific inhibitors, agonists, and antagonists. These studies are expected to lead to the development of new immunomodulating drugs. We recently proposed the extracellular ATP model of thymocyte differentiation and peripheral CTL and Th cell effector functions. This, in turn, suggested the possibility of modulating T cell development using ATP analogs. The focus of this project was to investigate the effects of ligands of purinergic receptors (extracellular ATP and adenosine) and their use for immunomodulation purposes. Different ATP analogs and inhibitors of extracellular protein phosphatases were tested using fetal organ thymus culture (FTOC) in vitro to model thymocyte differentiation in vivo. Dramatic changes in thymocyte subset composition were observed using ectophosphatase inhibitors and purinergic receptor ligands. Incubation with ectophosphatase inhibitors (microcystin) caused strong blocking of differentiation from DN to DP subsets with no major changes in the proportion of CD8+ SP and CD4+ SP thymocytes, suggesting that the phosphorylation of ectodomains may be involved in cell-cell interactions leading to thymocyte differentiation. In the presence of ATP, there was a dramatic block of differentiation from DN to DP thymocytes. Slowly- hydrolyzable ATP analogs had effects similar to ATP0. Analysis of these experiments supports the view that the major mechanistic components of effects of ATP on thymocyte differentiation in FTOC is transmembrane signaling through purinergic receptors, while processes of ectophosphorylation partially contribute to overall effect. It is concluded that the precise identification of purinergic receptor subclasses involved in thymocyte differentiation using different ATP analogs is not possible, due to the length of the FTOC; however, the observed differences between the effects of adenosine and ATP analogs suggest a differential role for P1 and P2 classes of receptors. Taken together, these studies point to the ATP analogs as promising drugs for immunomodulation.